Hepatitis C virus (HCV) is the major cause of transfusion and community-acquired non-A, non-B hepatitis worldwide. Approximately 2% of the world's population are infected with the virus. In the Unites States, hepatitis C represents approximately 20% of cases of acute hepatitis. Unfortunately, self-limited hepatitis is not the most common course of acute HCV infection. In the majority of patients, symptoms of acute hepatitis resolve, but alanine aminotransferase (a liver enzyme diagnostic for liver damage) levels often remain elevated and HCV RNA persists. Indeed, a propensity to chroninicity is the most distinguishing characteristic of hepatitis C, occurring in at least 85% of patients with acute HCV infection. The factors that lead to chronicity in hepatitis C are not well defined. Chronic HCV infection is associated with increased incidence of liver cirrhosis and liver cancer. No vaccines are available for this virus, and current treatment is restricted to the use of alpha interferon, which is effective in only 15-20% of patients. Recent clinical studies have shown that combination therapy of alpha interferon and ribavirin leads to sustained efficacy in 40% of patients (Poynard, T. et al. Lancet 1998, 352, 1426-1432.). However, a majority of patients still either fail to respond or relapse after completion of therapy. Thus, there is a clear need to develop more effective therapeutics for treatment of HCV-associated hepatitis.
HCV is a positive-stranded RNA virus. Based on comparison of deduced amino acid sequence and the extensive similarity in the 5′ untranslated region, HCV has been classified as a separate genus in the Flaviviridae family, which also includes flaviviruses such as yellow fever virus and animal pestiviruses like bovine viral diarrhea virus and swine fever virus. All members of the Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known virus-specific proteins via translation of a single, uninterrupted, open reading frame.
Considerable heterogeneity is found within the nucleotide and encoded amino acid sequence throughout the HCV genome. At least six major genotypes have been characterized, and more than 50 subtypes have been described. The major genotypes of HCV differ in their distribution worldwide, and the clinical significance of the genetic heterogeneity of HCV remains elusive despite numerous studies of the possible effect of genotypes on pathogenesis and therapy.
The RNA genome is about 9.6 Kb in length, and encodes a single polypeptide of about 3000 amino acids. The 5′ untranslated region contains an internal ribosome entry site (IRES), which directs cellular ribosomes to the correct AUG for initiation of translation. As was determined by transient expression of cloned HCV cDNAs, the precursor protein is cotranslationally and posttranslationally processed into at least 10 viral structural and nonstructural (NS) proteins by the action of a host signal peptidase and by two distinct viral proteinase activities. The translated product contains the following proteins: core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
The N-terminal portion of NS3 functions as a proteolytic enzyme that is responsible for the cleavage of sites liberating the nonstructural proteins NS4A, NS4B, NS5A, and NS5B. NS3 has further been shown to be a serine protease. Although the functions of the NS proteins are not completely defined, it is known that NS4A is a protease cofactor and NS5B is an RNA polymerase involved in viral replication. Thus agents that inhibit NS3 proteolytic processing of the viral polyprotein are expected to have antiviral activity.
There are several patents which disclose HCV NS3 protease inhibitors. WO98/17679 describes peptide and peptidomimetic ihibitors with the following formula: U-E8-E7-E6-E5-E4-NH—CH(CH2G1)-W1, where W is one of a variety of electrophilic groups, including boronic acid or ester. E4 represents either an amino acid or one of a series of peptidomimetic groups, the sythesis of which are not exemplified. The lactam inhibitors described in the present case are not covered.
WO98/22496 discloses peptide inhibitors of the following general formula: R9—NH—CH(R8)—CO—NH—CH(R7)—CO—N(R6)—CH(R5)—CO—NH—CH(R4)—CO—N(R3)—CH(R2)—CO—NH—CH(R1)—E where E either an aldehyde or a boronic acid. R1 represents lower alkyl (optionally substituted by halo, cyano, lower alkylthio, aryl-lower alkylthio, aryl or heteroaryl), lower alkenyl or lower akynyl.
Llinas-Brunet, Bailey et al WO99/07734 have described hexa- to tetra-peptide analogs containing a P1 electrophilic carbonyl group, a phosphonate ester, or an aza-aminoacid analog. Also, Llinas-Brunet, Poupart et al. WO99/07733 describe peptides terminating in a carboxylate. This latter group of compounds are similar to those described by Steinkuhler et al. Biochemistry 37, 8899-8905 (1998) and Ingallinella et al. Biochemistry 37, 8906-8914 (1998). These investigators report that hexapeptide substrate hydrolysis products are inhibitors of HCV protease. For example, Ac-Asp-Glu-Dpa-Glu-Cha-Cys-OH (SEQ. ID. NO.:1) is reported to have a Ki of <1.0 μM. In related disclosures, Ac-Asp-(D)Asp-Ile-Val-Pro-Cys-OH (SEQ. ID. NO.:2) has been shown to be more effective than its all “L” isomer Llinas-Brunet et al. Bioorg. Med. Chem. Lett. 8 1713-1718 (1998).
Additional peptide inhibitors of HCV protease have been disclosed. Hart et al WO9846630 have described hepta-peptide analogs containing an ester linkage at the scissile bond. Zhang et al. WO9743310 discloses high molecular weight peptide inhibitors. These compounds are also distinct from the present inventions.
Based on the large number of persons currently infected with HCV and the limited treatments available, it is desirable to discover new inhibitors of HCV NS3 protease.